Project Summary The parasite Toxoplasma gondii can cause severe disease in immunocompromised patients and fetuses. In intestinal epithelial cells of cats, its definitive host, Toxoplasma converts into merozoites, which are an asexual form that ultimately generates gametes that upon fusion will form hundreds of millions of oocysts. These oocysts are shed within cat feces, are highly stable in the environment, extremely resistant to inactivation procedures, and even a single oocyst is infectious. Therefore the generation of oocysts plays a crucial role in the prevalence of toxoplasmosis. Despite the crucial epidemiological role of the cat intestinal stages they are the least characterized, mainly because these stages are not cultivatable in vitro and difficult to access in vivo. Our overall goal is to identify and characterize the Toxoplasma regulatory factors that drive the development of the initial cat intestinal stages. We hypothesize that by upregulating multiple Toxoplasma regulatory factors that are normally only expressed in the cat intestine we can learn which factors play an important role in Toxoplasma conversion into the initial merozoite stage. In our first aim we will develop 15 transgenic parasite lines in each of which we can inducibly express a key Toxoplasma regulatory factor in vitro, which is normally only expressed in the cat intestine. Upon expression of these regulatory factors we will assess if they induce expression of genes that are normally only expressed in the cat intestine using high- throughput RNA sequencing. In our second aim we will test the hypothesis that the combined upregulation of multiple regulatory factors characterized in Aim 1 can convert Toxoplasma to merozoites. We will transiently transfect a merozoite reporter parasite line with a pool of all the regulatory factors from Aim 1 cloned behind a constitutive promoter. Our reporter parasites will express a drug-resistance and fluorescence marker driven by a merozoite promoter allowing us to visualize and select parasites that have successfully switched to merozoites. We will determine the relative frequency of parasites that successfully convert into merozoites. We will subsequently investigate the effect of individual factors, by withdrawing them from the transfection pool, on the formation of merozoites. This will identify what factors are essential for the stage conversion process. We will perform transcriptional profiling to confirm the parasite has converted to merozoites. If the complete pool of regulatory factors together does not induce merozoite stage conversion we will add small molecules that target histone modifying enzymes and simulate a more cat-like gut environment by infecting polarized cat epithelial cells with the transfected parasites. We will also perform a small CRISPR/CAS9 screen to identify putative repressors of merozoite stage conversion. This high risk - high reward exploratory R21 study uses innovative approaches that have the potential to discover how to culture merozoites in vitro, which could eventually contribute to the development of novel transmission blocking drugs or vaccines against Toxoplasma.